The present invention relates immunologically active peptides derived from a novel retrovirus of the HIV group, MVP5180/91. The invention further relates to the use of these peptides in diagnostic compositions and as immunogens.
Retroviruses which belong to the HIV group give rise, in humans infected with them, to disease symptoms which are summarized under the collective term immune deficiency or AIDS (acquired immune deficiency syndrome). Epidemiological studies demonstrate that the human immunodeficiency virus (HIV) represents the etiological agent for the overwhelming majority of AIDS cases. A retrovirus which was isolated from a patient and characterized in 1983 was given the designation HIV-1 (Barrxc3xa9-Sinoussi, F. et al., Science 220: 868-871 (1983)). A variant of HIV-1 is described in WO 86/02383.
Until 1993, the known HIV-1 isolates were categorized into the five subtypes A-E on the basis of sequence comparisons and epidemiological standpoints (G. Myers et al., Human Retroviruses and AIDS 1992. xe2x80x9cA compilation and analysis of nucleic acid and amino acid sequences.xe2x80x9d Los Alamos Laboratory, Los Alamos, USA (1992)).
A second group of human immunodeficiency viruses was identified in West Africa in 1985 (Clavel, F. et al., Science 233: 343-346 (1986) and designated human immunodeficiency virus type 2 (HIV-2) (EP-A-0 239 425). While HIV-2 retroviruses clearly differ from HIV-1, they also are related to monkey SIV immunodeficiency viruses. Like HIV-1, HIV-2 also gives rise to AIDS symptoms.
EP-A-0 345 375 describes another variant of an immunodeficiency retrovirus, which is designated HIV-3 retrovirus (ANT 70). The isolation of a different variant of immunodeficiency virus is also described in Lancet 340: 681-682 (1992).
Human immunodeficiency viruses characteristically exhibit a high degree of variability which significantly complicates attempts to compare the different isolates. For example, when comparing diverse HIV-1 isolates, high degrees of variability occur in some regions of the genome while other genome regions are comparatively well conserved (Benn, S. et al. Science 230: 949-951 (1985)). A substantially greater degree of polymorphism also has been observed in HIV-2 (Clavel, F. et al., Nature 324: 691-695 (1986)). The highest degree of genetic stability is possessed by regions in the gag and pol genes, which encode proteins which are structurally and enzymatically essential; some regions in the env gene, and also the genes (vif, vpr, tat, rev and nef) which encode regulatory proteins, exhibit a high degree of variability. In addition, it has also been demonstrated that antisera against HIV-1 also cross-react with gag and pol gene products from HIV-2 although only a low degree of sequence homology was present. These two viruses also did not hybridize with each other to any significant extent unless conditions of very low stringency were used (Clavel, F. et al., Nature 324: 691-695 (1986)).
In view of the wide dissemination of the retroviruses of the HIV group, and to the fact that there is a period lasting from a few to many years (2-20) between the time of infection and the time at which unambiguous symptoms of pathological changes are recognizable, it is of great importance epidemiologically that infection with retroviruses of the HIV group be detected as early as possible and, in particular, in a reliable manner. This is not only of importance when diagnosing patients who exhibit signs of immune deficiency, but also for screening blood donors. However, antibodies cannot be detected, or can be detected only weakly, in some sera when retroviruses of the HIV-1 or HIV-2 type, or constituents of these viruses, are used in detection systems. This is true even though the patients from which the sera are derived exhibit signs of immune deficiency. Thus, a need exists for a better method for detecting HIV infection, which does not use the previously known HIV-1 or HIV-2 type antigens.
Recently, another retrovirus that causes immune deficiency has been discovered. MVP5180/91 was isolated in 1991 from the peripheral lymphocytes of a 34-year old female patient from the Cameroons who exhibited signs of immune deficiency. This retrovirus originates geographically from a region in Africa which is located between West Africa, where infection with HIV-1 and HIV-2 viruses is endemic, and East Africa, where it is almost exclusively HIV-1 which is present. DE 43 18 186 describes nucleotide sequences from the viral genome of MVP5180/91 and amino acid sequences deduced therefrom. This retrovirus has been deposited, in accordance with the terms of the Budapest Treaty, in the European Collection of Animal Cell Cultures (ECACC) under the number V 920 92 318.
Similar to HIV-1 and HIV-2, MVP5180/91 grows in the following cell lines: HUT 78, Jurkat cells, C8166 cells and MT-2 cells. The isolation and multiplication of viruses are described in detail in Viral Quantitation in HIV Infection, Jean-Marie Andrieu (Ed.), John Libbey Eurotext (1991). The procedures described in that publication are incorporated herein by reference.
MVP5180/91 possesses a magnesium-dependent reverse transcriptase, which is not manganese-dependent. This represents a further feature possessed in common with the HIV-1 and HIV-2 viruses.
While anti-env antibodies in sera from German patients who are exhibiting signs of immune deficiency are weakly detected using the virus MVP5180/91, the sera react strongly when an HIV-1 virus is used instead of MVP5180/91 (DE 43 18 186). This stronger detection reaction was located principally in the gp41 protein. Thus, MVP5180/91 and HIV-1 are immunologically distinct.
The reliable detection of HIV infection is of particular interest today in connection with blood donation. In relation to ensuring the viral safety of blood and blood products, the immunochemical testing of individual donations in blood banks for HIV-1 antibodies became obligatory once specific anti-HIV-1 tests became available in 1985. After HIV-2 had been discovered in 1986, it became clear that it was not possible to detect HIV-2-specific antibodies as reliably with established HIV-1 tests as it was to detect anti-HIV-1 using corresponding HIV-1 antibody tests. Since 1989, xe2x80x9ccombination testsxe2x80x9d have been available which permit the simultaneous, non-differentiating, detection of anti-HIV-1 and anti-HIV-2. The majority of commercially available anti-HIV-1/anti-HIV-2 combination tests are based on HIV antigens which have been prepared recombinantly or by peptide synthesis.
Whereas the use of HIV-1 and HIV-2 antigens in the diagnosis of retrovirus infection is well-known, the diagnostic significance of the peptides from MVP5180/91 have thus far not been determined.
It is therefore an object of the present invention to provide an immunologically active peptide comprising at least 15 consecutive amino acids selected from the amino acids in the following sequence (SEQ ID NO:1):
VWGIRQLRARLQALETLIQNQQRLNLWGXKGKLIXYTSVKWNTSWSGR,
wherein X is C or S. This peptide detects antibodies against retroviruses of the HIV type.
The invention further relates to a kit for detecting antibodies against viruses which cause immune deficiency comprising the above described peptide.
The invention further relates to a diagnostic agent for detecting an antibody against a retrovirus that causes immune deficiency, the diagnostic agent comprising the above described peptide and a detectable label that is capable of detecting the binding of the peptide with the antibody.
In another embodiment, the invention relates to a method of detecting the presence of anti-retrovirus antibodies in a sample, the method comprising contacting the sample with the above described diagnostic agent and detecting the presence of antibody bound to the diagnostic agent as a result of the contacting.
Another embodiment of the invention relates to an immunogen comprising (a) an amount of the above described peptide and (b) a physiologically-acceptable excipient therefor, wherein said amount is sufficient to elicit an immune response that is protective of a susceptible mammal against retrovirus infection.
In another embodiment, the invention relates to a method of immunizing a mammal against retrovirus infection, comprising administering to the mammal an effective amount of the above described immunogen.
Another embodiment of the present invention relates to an isolated DNA molecule which encodes the above described peptide.
Another embodiment relates to a method of detecting in a sample nucleic acids encoding a retrovirus that causes immune deficiency, comprising the steps of: (a) hybridizing a labeled DNA molecule to nucleic acids encoding a retrovirus in said sample, wherein said labeled DNA molecule is prepared by labeling the above described DNA molecule with a detectable label, and (b) detecting the hybridizing by means of said detectable label.
In another embodiment, the invention relates to a method of detecting in a sample nucleic acids encoding a retrovirus that causes immune deficiency, comprising subjecting said nucleic acids to a Polymer Chain Reaction (PCR), wherein the PCR employs at least two oligonucleotide primers that anneal to a nucleic acid encoding a retrovirus that causes immune deficiency, wherein one of the primers is complementary to a first nucleotide sequence comprising the sequence of the above described DNA molecule, or its complementary sequence, wherein the other primer is complementary to a second nucleotide sequence comprising a nucleic acid molecule encoding a retrovirus that causes immune deficiency, whereby a geometrically amplified product is obtained only when the first and second nucleotide sequences occur within the same nucleic acid molecule encoding a retrovirus that causes immune deficiency.